An Optimized Method For High-titer Lentivirus Preparations Without Ultracentrifugation

Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells.Currently,most protocols for generating high-titer lentivirus require ultracentrifugation,which can be an instrumental barrier for routine operations in a laboratory.The HEK293T cells were transfected with a packaging vector expressing a green fluorescent protein?GFP?along with transfer and envelope vectors,and the virus-containing medium being collected at 48 hours after transfection was used as the starting materials for the concentration.To measure the titer of the functional virus,the concentrated virus was added to the HEK293T cells,and the percentage and relative intensity of the GFP-positive cells were measured with a flow cytometer.We used a20%sucrose gradient centrifugation for 4 hours,which is most commonly used in published protocols,as the starting point.The concentration of functional virion particles,reflected as the percentage of the cells infected,was increased in a linear relationship with RCF in the range of 2,000–10,000 g.The recovery yield of 10,000 g centrifugation was 85.6?0.07%.Intriguingly,10,000 g centrifugation showed a 185.8?23.7%transduction efficiency compared with the ultracentrifuge at the speed of 90,000g for 90 minutes with otherwise identical settings.In the concentration window between5%and 50%of sucrose,this purification method showed reliable performance in enriching the GFP-expressing lentivirus and a more detailed analysis showed that 10%of sucrose exhibited the best effect in the virus concentration.The change in the virus titer upon prolonged storage of the lentivirus at 4?C was tested.The infection efficiency of the un-concentrated and concentrated viruses was reduced exponentially??=1.4days and 1.3 days,respectively?.And the infection efficiency after one round of freeze and thaw was reduced by 33.3?6.4%??=1.1 rounds?The hippocampal neuronal cultures were infected with 4?l of the virus?2.04?10⁷TU/ml?.almost 100%of the neurons showed GFP fluorescence at days in vitro?DIV?14 when the concentrated virus was added at DIV 4.The miniature excitatory postsynaptic synaptic currents?mEPSCs?and miniature inhibitory postsynaptic synaptic currents?mIPSCs?of the infected neurons were measured and compared with the uninfected neurons.the mEPSCs and mIPSCs were normal both in frequencies and in amplitudes,implying that the addition of the concentrated virus did not cause detectable toxic effects to the neurons.In addition,the miniature synaptic transmission from the neurons infected with purified lentivirus at DIV 10 was measured when the synapses had already formed.The results show that the virus infection had no effect on the synaptic transmission,implying that the neurons in the late development stage?DIV 10?were not affected by the virus infection.Using a stereotaxic injection,the concentrated virus was injected into the CA1 and CA3 regions of the hippocampus in newborn mice pups.The GFP-positive CA1 and CA3 neurons were observed at P12,demonstrating the in vivo application of the method.To further test whether ot not the injected virus might result in a significant immune response,we detected the GFAP-postive astrocytes on hippocampal tissue slides from mice injected unilaterally with the lentivirus purified with 10,000 g centrifugations.The results show that no significant aggregation of GFAP-positive astrocytes were observed around the virus-injected sites in CA3 or CA1region of hippocampus comparing to the uninjected hippocampus.This method can be easily modified for the large-scale preparation of lentiviral stock.And the titer of the concentrated virus is 2.00?10⁸ TU/ml at a 1:500concentration ratio.And double concentrated virus reach at a 1:600 concentration ratio.In summary,a relatively simple and practical method to purify a high-titer and high-quality lentivirus for both in vitro and in vivo applications has been described.